Nontypeable Haemophilus influenzae (NTHi) commonly colonize the mucosal surfaces of the human upper respiratory tract. For the most part, this species resides in that environment in a benign (symbiotic?) relationship with the human host. With pathophysiologic changes (obstruction of outflow ducts, loss of the mucociliary elevator, viral infection, etc.) in the airway anatomy and mucosal surface, NTHi can cause human upper and lower tract respiratory infection. Recent studies in our laboratory using established assays suggest that NTHi produces a biofilm and that a CMP-NANA synthase (siaB) and an (alpha2-3 sialyltransferase (siaA) mutant cannot make a biofilm. Our studies have also shown that both mutants can initially adhere to human bronchial epithelial cells as well as the NTHi 2019 parent strain. In addition, a mutation in another glycosyltransferase, Rfe, which is a homologue of an undecaprenyl-phosphate alpha-N-acetylglucosaminyltransferase, produces no biofilm. In addition, growth of NTHi 2019 in sialic acid deficient defined media significantly reduces biofilm formation. A mutation in lsgG, a homolog of the regulator, Escherichia coli modE, also produces a reduced amount of biofilm. Formation. Our studies would suggest that NTHi makes a biofilm matrix in part composed of sialic acid and lacks glucose and galactose. Based on these observations, we propose to explore the factors controlling the production of biofilm formation during NTHi infection of bronchial epithelia, the impact of biofilm formation on LOS expression and interactions between NTHi and primary human bronchial epithelial cells during long-term infections. The hypotheses underlying this proposal are 1) that NTHi exists in a biofilm during bronchial infection and that this capability is under the control of a bacterial or environmental signal 2) that the NTHi biofilm is either entirely or predominately composed of carbohydrate, likely containing sialic acid and 3) that gene and protein expression of NTHi in the biofilm and planktonic phase are different, and 4) that there is a linkage between LOS expression and biofilm formation. To examine these hypotheses, we would propose the following Specific Aims: 1) The elucidation of the structure of the NTHi biofilm and the genes responsible for its biosynthesis, 2) The determination of the genes involved in biofilm biosynthesis and the differences in gene and protein expression in biofilm and planktonic NTHi and 3) The molecular analysis of the effects of prolonged NTHi infection on primary human bronchial epithelial cells.